Abstract Abnormalities in triglyceride (TG) metabolism are a critical risk factor for fatty liver disease, atherosclerosis, obesity and diabetes, all of which are major clinical problems worldwide. ApoAV is a new member of the apolipoprotein family and is synthesized exclusively in the liver. A fact that is often neglected is that the concentration of apoAV in human plasma is extremely low (114-258 ng/mL), 1,000 to 10,000-fold lower than that of apoB100 and apoAI, respectively. It has fascinated investigators for many years how a very low circulating level of apoAV can exert such a profound effect on plasma TG homeostasis in humans and animals. Because ~20% of apoAV is secreted into the circulation, its concentration is significantly higher in the liver than in plasma. Our preliminary data show that apoAV plays a critical role in regulating hepatic TG metabolism, and ethanol disrupts hepatic TG homeostasis and exacerbates the accumulation of excess TG in the liver, leading to more severe alcoholic liver disease (ALD) in apoAV knockout (KO) mice than in wild-type (WT) mice. We further found that ethanol inhibits expression of apoAV in a dose- dependent manner in the mouse primary hepatocytes. Chronic and binge ethanol feeding (i.e., the NIAAA model) for 4 weeks leads to a significant and rapid development of liver steatosis, gradually evolving from simple steatosis to alcoholic steatohepatitis and then to liver fibrosis in apoAV KO, but not WT mice. More importantly, ethanol significantly increases hepatic concentrations of lysophosphatidylcholine (lysoPC), a major fatty acid metabolite, by enhancing its biosynthesis in apoAV KO mice. A recent human study has found that lysoPC is a new and important biomarker for excess alcohol intake; however, its vital role in the pathogenesis of ALD is still unknown. By contrast, transgenic expression of the human APOAV gene in mice protects against ethanol-induced liver injury. Our results demonstrate that the absence of apoAV is an important risk factor for ALD and apoAV is a novel target for the prevention and the treatment of ALD. Thus, our preliminary results support the hypothesis that adeno-associated-virus 2/8 (AAV2/8)-mediated gene transfer of the human APOAV protects against ALD in ethanol-fed mice. Of the commonly used viral vectors, AAV produces the lowest immune response and is non-pathogenic even in the wild-type state, as well as is the most suitable viral vector for therapeutic applications. AAV2/8 (i.e., AAV-8 pseudotypes in which AAV-2 genomes are packaged into AAV-8 capsids) displays highly efficient liver gene transfer. Therefore, AAV2/8 is a promising method for gene therapy of ALD. We will test the hypothesis in two specific aims: First, we will investigate whether overexpression of APOAV protects against ethanol-induced liver steatosis in mice transduced with AAV2/8- APOAV. Second, we will explore whether AAV2/8-APOAV treatment prevents ethanol-triggered progression from simple steatosis to alcoholic steatohepatitis through lysoPC in mice. The proposed studies are innovative because this project may provide an efficacious strategy for the gene therapy of ALD.